The importance of coordinating cell growth with proliferation has been recognized for a long time. The molecular basis of this relationship, however, is poorly understood. Here we show that the ribosomal protein L23 interacts with HDM2. The interaction involves the central acidic domain of HDM2 and an N-terminal domain of L23. L23 and L11, another HDM2-interacting ribosomal protein, can simultaneously yet distinctly interact with HDM2 together to form a ternary complex. We show that, when overexpressed, L23 inhibits HDM2-induced p53 polyubiquitination and degradation and causes a p53-dependent cell cycle arrest. On the other hand, knocking down L23 causes nucleolar stress and triggers translocation of B23 from the nucleolus to the nucleoplasm, leading to stabilization and activation of p53. Our data suggest that cells may maintain a steady-state level of L23 during normal growth; alternating the levels of L23 in response to changing growth conditions could impinge on the HDM2-p53 pathway by interrupting the integrity of the nucleolus.The tumor suppressor protein p53 plays a pivotal role in preventing damaged and abnormal cells from becoming malignant, and its loss of function is associated with a majority of human cancers (26,36,37). The activity of p53 is not required for normal cell growth, and the protein is kept at low levels and inactive. This is accomplished by the proto-oncoprotein HDM2 (human counterpart of MDM2 in mice) through either ubiquitin-dependent p53 degradation in the cytoplasm (6, 7, 11) or repression of p53 transcriptional activity in the nucleus (17, 35). The HDM2 gene can, in turn, be transcriptionally activated by p53, constituting a feedback regulatory loop (1, 40). The current understanding of the major mechanisms of p53 activation includes one that is triggered by DNA damage and induces p53 phosphorylation through a cascade of protein kinases (5, 15, 32) and one that is triggered by aberrant oncogenic signals and is mediated by p14 ARF (p19 ARF in mice) (31). Little is known about the connection between p53 and ribosomal biogenesis, even though ribosomal biogenesis occupies the major part of a cell's life cycle. Whether and how p53 may be involved in checking ribosomal stress and the integrity of ribosomal biogenesis remains elusive (28).Previous studies have identified the interaction of the ribosomal protein L5 with HDM2 (14). Recently, it has been shown that the ribosomal protein L11 also interacts with HDM2, and through this interaction, L11 stabilizes and activates p53 and induces a cell cycle arrest (13,41). This is achieved, at least in part, through L11 inhibiting the E3 ligase activity of HDM2 (41). Based on the evidence that low levels of actinomycin D, which selectively inhibits rRNA transcription, enhances L11-HDM2 interaction, it has been proposed that L11 plays a role in the response to ribosomal perturbations to induce p53 and cell cycle arrest. In this study, we describe functional interactions of HDM2 with L23, a protein component in the 60S large ribosomal subunit. The...

As a shuttling protein, p53 is constantly transported through the nuclear pore complex. p53 nucleocytoplasmic transport is carried out by a bipartite nuclear localization signal (NLS) located at its C-terminal domain and two nuclear export signals (NES) located in its N-and C-terminal regions, respectively. The role of nucleocytoplasmic shuttling in p53 ubiquitination and degradation has been a subject of debate. Here we show that the two basic amino acid groups in the p53 bipartite NLS function collaboratively to import p53. Mutations disrupting individual amino acids in the NLS, although causing accumulation of p53 in the cytoplasm to various degrees, reduce but do not eliminate the NLS activity, and these mutants remain sensitive to MDM2 degradation. However, disrupting both parts of the bipartite NLS completely blocks p53 from entering the nucleus and causes p53 to become resistant to MDM2-mediated degradation. Similarly, mutations disrupting four conserved hydrophobic amino acids in the p53 C-terminal NES block p53 export and prohibit it from MDM2 degradation. We also show that colocalization of a nonshuttling p53 with MDM2 either in the nucleus or in the cytoplasm is sufficient for MDM2-induced p53 polyubiquitination but not degradation. Our data provide new insight into the mechanism and regulation of p53 nucleocytoplasmic shuttling and degradation.The tumor suppressor protein p53 plays a pivotal role in preventing damaged or abnormal cells from becoming malignant, and its loss of function is associated with a majority of human cancers (23). p53 activity is not required during normal cell growth, and the protein must be kept at low levels and inactive. This is accomplished by the proto-oncoprotein MDM2, either through ubiquitin-dependent p53 degradation in the cytoplasm (11,13,15) or repression of p53 transcriptional activity in the nucleus (19,30). The MDM2 gene is in turn transcriptionally activated by p53, constituting a feedback regulatory loop (2,33).Growing evidence has shown that p53 is regulated primarily by its protein stability (1, 31). A major mechanism of p53 stabilization and activation is triggered by DNA damage, which induces protein phosphorylation. Through a cascade of activity of protein kinases, DNA damage, such as that caused by ionizing radiation, induces multiple-site phosphorylation of p53 at its N and C termini. Phosphorylation of the N terminus of p53 affects its affinity for MDM2 and subsequent degradation (10,18,25). A recent study demonstrates that phosphorylation at the p53 N terminus inhibits its nuclear export, underscoring the importance of export in controlling p53 function (38). Another mechanism of p53 stabilization is triggered by aberrant growth signals, such as oncogenic Ras or Myc, and is mediated by a small protein called p14 ARF (p19 ARF in the mouse) encoded by DNA at the p16 INK4a locus (24). Upon induction, ARF binds to MDM2 (21, 39), inhibits its E3 ligase activity (14), and leads to p53 stabilization. This is achieved, at least in part, by blocking the nuclear...

A new bidirectional laser Doppler velocimeter (LDV) is described for absolute measurement of the speed of red blood cells flowing in individual retinal vessels. The basic component of the instrument is a standard retinal camera that eliminates the need for a contact lens. The laser beam is delivered to the eye through the fundus illumination optical system of the camera. Target fixation is done with the eye under examination. The measurements are independent from the ocular refraction; only the axial length of the eye need be determined. The instrument markedly simplifies the technique of retinal blood flow measurement.

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